The autoantibody response in systemic autoimmune diseases focuses on a specific set of autoantigens, but it is not clear why. At the outset of the current funding cycle, the Rothstein and Shiomchik labs had demon[unreadable] strated that activation of rheumatoid factor (RF)-specific B cells by their antigenic immune-complexes was greatly potentiated if chromatin was contained in these complexes, and this was dependent on intact Toll-like receptor (TLR) signaling, most likely via TLR9. These in vitro findings led to the central hypothesis that cer[unreadable] tain autoanfigens (particularly chromafin) can be ligands for TLRs or other innate immune response recep[unreadable] tors, and that the ligation of such receptors can play a crifical role in autoimmune responses of B cells. In the last period we, along with the other PPG invesfigators, demonstrated that indeed TLRs regulate autoimmu[unreadable] nity in vivo, a concept also corroborated by others in several systems. We showed that TLR9 was required for anti-DNA type ANAs and extended the paradigm to TLR7, showing that RNA-associated autoantibodies required this molecule. Together these discoveries have led to a new understanding ofthe pathogenesis of autoimmune diseases, as well as treatment approaches, some of which is already reaching the clinic. Though we found that TLR7 (and MyD88) were proinflammatory, we unexpectedly found that TLR9 para[unreadable] doxically suppressed global disease. These findings raise two major unanswered quesfions: a) how does TLR9 play a regulatory role in disease;and b) are TLR7 and TLR9 the only innate-immune sensing receptors of relevance in systemic autoimmunity? We will address these questions using in vivo genetic and in vitro biochemical approaches. In Aim 1 we will test the hypothesis that TLRs have tissue-specific functions in vivo, in part explaining the effect of deletion of TLR9 in all tissues. In Aim 2 we will test the hy[unreadable] pothesis that TLR9 overexpression[unreadable]either globally, or in specific cell types[unreadable]^will suppress some or all as[unreadable] pects of disease, thus highlighfing a therapeufic approach. In Aim 3 we will use F2 genetic crosses to test the roles in disease of the remaining known innate immune nucleic acid sensing pathways. Aim 4 will addresses several hypotheses concerning interactions between TLR9 and TLR7, as our unpublished genetic evidence shows that disease exacerbafion due to TLR9 delefion requires TLR7. These studies should provide novel insights into how innate immune signaling regulates systemic autoimmune disease in vivo RELEVANCE (See instructions): This work will enable us to understand much better how the immune system attacks the body during autoimmune diseases like Systemic Lupus Erythematosus. Since these diseases are complex, we need animal models to understand how they work. We are using state ofthe art genetic tools to generate specific types of mutant mice to test how the two major limbs ofthe immune system, innate and adaptive, interact to cause autoimmune diseases.